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WO1996041529A1 - Compositions et procedes a utiliser pour lutter contre des populations d'insectes - Google Patents

Compositions et procedes a utiliser pour lutter contre des populations d'insectes Download PDF

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Publication number
WO1996041529A1
WO1996041529A1 PCT/US1996/008027 US9608027W WO9641529A1 WO 1996041529 A1 WO1996041529 A1 WO 1996041529A1 US 9608027 W US9608027 W US 9608027W WO 9641529 A1 WO9641529 A1 WO 9641529A1
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Prior art keywords
insect
larvae
acmnpv
agent
zea
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PCT/US1996/008027
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English (en)
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Jan Oliver Washburn
Loy Elaine Volkman
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The Regents Of The University Of California
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Priority to AU58843/96A priority Critical patent/AU5884396A/en
Publication of WO1996041529A1 publication Critical patent/WO1996041529A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/50—Isolated enzymes; Isolated proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/14011—Baculoviridae
    • C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
    • C12N2710/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • This invention relates generally to the fields of biology and chemistry. More particularly, the present invention relates to compositions and methods for use in control (e.g., reduction or elimination) of insect populations.
  • Autographa califomica M Nucleopolyhedrosis Virus is the best studied of the baculoviruses and is known to infect the larvae of approximately 30 species in several families within the insect order, Lepidoptera [Granados, R. R., and Williams, K. A. (1986). In vivo infection and replication of baculoviruses. In The biology of baculoviruses (eds. R. R. Granados and B. A. Federici) vol. I, CRC Press, Inc., Boca Raton, FL].
  • Virus Taxonomy sixth report of the international committee on taxonomy of viruses.
  • Nucleocapsids enter differentiating and mature columnar cells in the midgut epithelium following fusion of the viral envelope with the microvillar membrane of these cells [Kawanishi, C. Y., Summers, M. D., Stoltz, D.
  • Progeny nucleocapsids produced by infected midgut cells also bud from the basal plasma membrane containing a newly synthesized, viral-encoded glycoprotein, gp64 [Keddie, B. A., Aponte, G. W., and Volkman, L. E. 1989. The pathway of infection of Autographa californica nuclear polyhedrosis virus in an insect host. Science 243, 1728-1730] .
  • This budded virus (BV) spreads infection to the other tissues of the insect which produce both BV and polyhedra. Insects die after virtually all their tissues are infected massively, and cadavers liquefy releasing numerous polyhedra which horizontally transmit the virus to susceptible larvae.
  • compositions and methods for use in controlling insect populations wherein a first agent which suppresses insect immune function (as hereinafter defined) of a target insect is employed in conjunction with a second agent which is selected from the group consisting of insect pathogens (as hereinafter defined) for the target insect.
  • the first agent is a polypeptide having immunosuppressive activity in the target insect or a polynucleotide sequence encoding the polypeptide; in the latter case, the polynucleotide may be conveniently administered to the target insect in a vector comprising the second agent (e.g., AcMNPV or similar insect pathogen).
  • Fig. 1 illustrates the proportions of H. zea larvae expressing lacZ at various hpi (hours post inoculation) with 1100 polyhedra of
  • Fig. 2 illustrates comparisons of the proportions of lacZ expressing H. virescens and H. zea larvae and number of viral foci/infected insect sampled 24 to 40 hpi with 22 polyhedra of AcMNPV-hsp70/lacZ; and Fig. 3 illustrates the proportion of H. zea larvae expressing lacZ 48 hpi with 110 polyhedra of AcMNPV-hsp70/lacZ.
  • compositions and methods are provided for use in controlling insect populations.
  • the compositions of the present invention comprise in combination a first agent which suppresses insect immune function in an amount effective to suppress the immune function of a target insect and a second agent selected from the group consisting of insect pathogens in an amount effective to control or eliminate a population of the target insect.
  • the methods of the present invention comprise administering to a target insect population the first agent before, after or simultaneously with the second agent.
  • the first agent suppresses insect immune function of a target insect.
  • an agent which suppresses insect immune function may be defined as a composition which interferes with and prevents the completion of the encapsulation/melanization response which characterizes the known cellular immune response of insects to invading organisms or objects.
  • Such composition disables the cellular immune response and prevents the host insect from successfully eliminating a foreign body (e.g., an insect pathogen).
  • Organic or inorganic chemical immunosuppressants such as DDCA (diethylditbiocarbamic acid), PTU (phenylthiourea), benzamidine and other compounds [Saul, S.J. and Sugumaran, M. 1988. Prophenol oxidase activation in the hemolymph of Sarcophaga bullata larvae. Arch. Insect Biochem. and Physiol. 7:91-104] which interfere with components of the phenyloxidase system which is essential for successful activation and completion of the encapsulation response [Vinson, S. B. 1990. How parasitoids deal with the immune system of meir host: an overview. Arch. Insect Biochem. and Physiol. 13:3-27 1990], may suitably be employed as first agents in accordance with the present invention. Use of DDCA in conjunction with an insect pathogen is illustrated in the examples herein.
  • the first agent is a polypeptide having immunosuppressive activity in the target insect.
  • examples of such peptides include VH1.1 and related gene products in the cysteine-rich gene family [Dib-Hajj, S. D. , Webb, B. A. , and Summers, M. D. 1993. Structure and evolutionary implications of a "cysteine-rich" Campoletis sonorensis polydnavirus gene family. Proc. Natl. Acad. Sci. USA 90:3765-3769] of the Camplotetis sonorensis polydnavirus. Complete nucleotide sequences for the genes encoding VHl.
  • insect pathogen may be defined as an agent (such as a virus, bacterium, protozoan or fungus) which infects and causes disease symptoms in insects.
  • agent such as a virus, bacterium, protozoan or fungus
  • Exemplary of an insect pathogen known to infect about 30 species of lepidopterans is AcMNPV.
  • AcMNPV is the best studied of the baculoviruses, and its complete DNA sequence has been reported [Ayres, M.D., Howard, S.C., Kuzio, J., Lopez-Ferber, M. and Possee, R.D. 1994.
  • the first agent is a polypeptide having immunosuppressive activity in the target insect and is provided in the form of a polynucleotide sequence encoding the polypeptide.
  • the polynucleotide first agent is advantageously administered in accordance with this class of embodiments to the target insect in a vector comprising the second agent (e.g., AcMNPV or similar insect pathogen).
  • a set of genetically engineered recombinants of AcMNPV that expands the range of hosts that succumb to fatal viral infections.
  • These recombinants comprise at least one polynucleotide sequence encoding a polypeptide insect immunosuppressant.
  • optimum amounts of a composition in accordance with the present invention for use in controlling a population of a given target insect may readily be determined empirically by quantifying dosage-mortality relationships within target insect populations.
  • compositions of the present invention are administered to the target insect population in a manner known per se for administration of heretofore-known chemical and biological pesticides, for example by oral microinjection of inoculum in the laboratory or by spraying infested crop plants in the field.
  • the compositions of the present invention generally comprise a suitable carrier or excipient appropriate to the method of administration thereof, as would be readily appreciated by those working in the field.
  • H. zea Using the highly resistant and economically important host, Helicoverpa zea, as a model resistant species, a contributing factor to the physiological basis of viral resistance has been discovered and a means for circumventing it developed.
  • a reporter gene construct of AcMNPV the pathogenesis of viral infections in H. zea, an important agricultural pest reported to be highly resistant to infection by this baculovirus, has been examined. Specifically, it has been determined that H. zea larvae are actually very susceptible to initial infection by AcMNPV, but that the cellular immune response of H. zea is involved in stopping the spread and eliminating systemic AcMNPV infections. Further, it has been shown that down regulating the cellular immune response of H. zea results in the expansion of AcMNPV infections within host tissues.
  • H. zea larvae are readily infected by AcMNPV, recombinant viruses are able to express these genes and mereby disable the immune response allowing infections to expand throughout the host tissues and ultimately to kill the insect.
  • the early events during viral infection are very similar to those observed in a closely related but highly susceptible host, H. virescens.
  • the mechanism for resistance does not involve initial infection of the midgut epithelium following ingestion of virus; rather, an important mechanism contributing to viral resistance operates shortly after AcMNPV moves from the midgut epithelium to secondary target cells (i.e., tracheal epidermal cells of tracheae associated with the midgut).
  • secondary target cells i.e., tracheal epidermal cells of tracheae associated with the midgut.
  • Microscopic analyses of viral plaques within the tracheal epidermis show that shortly after systemic AcMNPV infections are established, the cellular immune system of H. zea larvae is activated, and hemocytes begin aggregating at these sites of infections. Subsequently, melanization and encapsulation of the viral plaques and a cessation of viral spread through the tracheal system and into the other host tissues were observed. Characteristics of encapsulated viral foci are identical to those previously described for the cellular insect defense response to other pathogens and parasites.
  • constructs of AcMNPV which contain polydnavirus genes which suppress me insect immune system are provided.
  • Polydnaviruses like AcMNPV, are double-stranded DNA viruses.
  • Polydnaviruses are obligate symbionts associated with the calyx cells of the reproductive tracts of parasitic wasps such as Camploetis sonorensis (Ichneumonidae) . They have a unique relationship both with the parasitic hymenoptera in which they are found and with the hosts that these wasps parasitize [Vinson, 1990, supra; Li, X. and Webb, B. A. 1994.
  • VHl.l a gene originating from Camplotetis sonorensis polydnavirus
  • the baculovirus was then used as a delivery system to test the hypothesis that the VHl.l interferes with the encapsulation response of H. virescens.
  • Experimental evidence demonstrated that encapsulation was significantly reduced relative to control insects [Li and Webb, 1994, supra]. Results from this study are significant, both in demonstrating an immunosuppressant function for the VHl.l gene product, and in showing that a recombinant of AcMNPV containing VH 1.1 expresses the gene product in amounts sufficient to suppress the cellular immune response of H. virescens.
  • AcMNPV is widely used as an expression vector in cell culture, and is employed herein in the examples. Nonetheless, it would be readily apparent to those working in the field that the general principles described herein would be applicable to other insect pathogens which may serve as vector for one or more immunosuppressive genes.
  • insect pathogens which might serve as vectors for use in accordance with the present invention include, but are not limited to, other baculoviruses and other microorganisms pathogenic to a target insect population.
  • This recombinant contains all the genes found in wild type virus plus the E. coli b-galactosidase gene driven by the Drosophila heat shock promoter (hsp70).
  • This reporter gene is expressed early in virus-infected cells [Engelhard, E. K., Kam-Morgan, L. N. W., Washburn, J. O., and Volkman, L. E. 1994.
  • the insect tracheal system a conduit for the systemic spread of Autographa californica M nuclear polyhedrosis virus. Proc. Natl. Acad. Sci. USA 91:3224-3227; Flipsen, J. T.
  • Polyhedra of AcMNPV-hsp70/lacZ were used to infect test larvae in all experiments described here. Polyhedra were first isolated from liquefied cadavers of T. ni and partially purified by sucrose gradient centrifugation [Summers, M. D. and Smith, G. E. (1987). A manual of methods for baculovirus vectors and insect cell culture procedures, Texas Agricultural Experiment Station Bulletin No. 1555 (Tex. Agric. Exp. Stn. , College Station, TX]. Pelleted polyhedra were suspended in a neutrally buoyant solution of glycerol and water (3:2 v/v). Viral inocula were quantified using a hemocytometer and held at 4° C until use.
  • Helicoverpa zea and Heliothis virescens larvae were provided by American Cyanamid Corporation, Princeton, NJ. Prior to experimental procedures, all larvae were reared in cohorts on a synthetic diet [Tanada, Y. and G. Y. Chang. 1968. Resistance of the alfalfa caterpillar, Colias eurytheme, at high temperatures to a cytoplasmic-polyhedrosis virus and thermal inactivation point of the virus. J. Invertebr. Pathol. 10:79-83; Keddie, B. A. and L. E. Volkman. 1985. Infectivity difference between the two phenotypes of Autographa californica Nuclear Polyhedrosis Virus: importance of the 64K envelope glycoprotein.
  • mice were inoculated orally using a microapplicator (ISCO) fitted with a plastic tuberculin syringe (1 cc) with a 32 gauge needle. During inoculations, a blunt-tip needle was inserted through the mouth and into the anterior region of the larval midgut where polyhedra were delivered. Within experiments, both the volume and dosage were held constant, but among experiments, the inoculum volume was varied from 0.5 to 2.0 ml and the dosage from 17 to 1100 polyhedra per insect. Following injection, all test insects were maintained in individual containers at 28 ⁇ 1 °C.
  • ISCO microapplicator
  • Pathogenesis was assessed by challenging 4° H. zea and H. virescens with the same inoculum of AcMNPV and subsequently comparing the proportions of infected insects and the number of viral foci per infected insect. Larvae were sacrificed at 24 and 36-40 hpi in order to assess AcMNPV distribution early in the systemic infection of hosts.
  • Results from this experiment clearly demonstrate that H. zea is actually very similar to H. virescens with regard to susceptibility to AcMNPV infection. Moreover, the results show that resistance to viral-induced mortality in H. zea does not involve the infection of the midgut epithelium; rather the mechanism of resistance operates after infections have entered the tracheal system of the host.
  • DDCA diethyldithio-carbamic acid
  • zea were inoculated with virus, injected intrahemocoelically with DDCA at 15 to 24 hpi and subsequently processed for viral signal at 48 hpi.
  • Fig. 3 Results comparing the proportions of insects signaling lacZ at 48 hpi are shown in Fig. 3. Each bar represents the proportion of signaling insects from a population of 7 to 12 insects. For both third and fourth instar larvae inoculated with virus and DDCA, greater proportions of larvae expressing lacZ were found compared to control insects. In addition, there were striking differences in the distribution and size of viral plaques in DDCA treated insects compared to controls. Among controls, individual viral foci were very small, each typically consisting of less than 10 infected tracheal epidermal cells; the largest plaques covered less than 5% of the tracheal vessels associated with the midgut and many larvae exhibited patches of melanization along infected tracheae.
  • H. zea is a suitable host for C. sonorensis, and C. sonorensis does in fact immunosuppress H. zea.
  • lacZ signal patterns in parasitized and unparasitized larvae confirms the role of the cellular immune system in stopping the spread and eliminating AcMNPV infections in this host. Specifically, a greater proportion of parasitized larvae expressing lacZ compared to unparasitized larvae is observed. In addition, the lacZ signal is more widely distributed in parasitized, virus-infected larvae compared to unparasitized, virus-infected larvae.

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Abstract

L'invention porte sur des compositions et des procédés à utiliser pour lutter contre des populations d'insectes, comprenant un premier agent qui supprime la fonction immunitaire propre à l'insecte chez un insecte cible combiné à un second agent qui est un pathogène pour l'insecte cible. Le premier agent est, de préférence, un polypeptide ayant une activité immunodépressive chez l'insecte cible ou une séquence polynucléotidique codant le polypeptide; dans le dernier cas, le polynucéotide peut être avantageusement administré à l'insecte cible dans un vecteur contenant le second agent (par exemple, le virus de la nucléopolydrose M Autographa californica (AAcMNPV ou un pathogène similaire).
PCT/US1996/008027 1995-06-12 1996-05-30 Compositions et procedes a utiliser pour lutter contre des populations d'insectes WO1996041529A1 (fr)

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AU58843/96A AU5884396A (en) 1995-06-12 1996-05-30 Compositions and methods for use in controlling insect popul ations

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US48952295A 1995-06-12 1995-06-12
US08/489,522 1995-06-12

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Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6355240B1 (en) 1995-07-26 2002-03-12 Basf Aktiengellschaft Enhanced insecticidal insect virus through the expression of heterologous proteins with early promoters
AUPP008097A0 (en) * 1997-10-29 1997-11-20 Luminis Pty Limited Insect immunoprotection and a pesticide
WO2010132097A2 (fr) * 2009-05-11 2010-11-18 Massachusetts Institute Of Technology Compositions et procédés de lutte contre des organismes nuisibles
CN113598134A (zh) * 2021-07-30 2021-11-05 云南省烟草公司昆明市公司 一种夜蛾黑卵蜂低温保藏的方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4879236A (en) * 1984-05-16 1989-11-07 The Texas A&M University System Method for producing a recombinant baculovirus expression vector
US5011685A (en) * 1988-04-06 1991-04-30 Boyce Thompson Institute For Plant Research, Inc. Baculovirus proteins and viral pesticides containing same
US5162222A (en) * 1989-07-07 1992-11-10 Guarino Linda A Use of baculovirus early promoters for expression of foreign genes in stably transformed insect cells or recombinant baculoviruses

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4879236A (en) * 1984-05-16 1989-11-07 The Texas A&M University System Method for producing a recombinant baculovirus expression vector
US5011685A (en) * 1988-04-06 1991-04-30 Boyce Thompson Institute For Plant Research, Inc. Baculovirus proteins and viral pesticides containing same
US5162222A (en) * 1989-07-07 1992-11-10 Guarino Linda A Use of baculovirus early promoters for expression of foreign genes in stably transformed insect cells or recombinant baculoviruses

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GAUGLER et al., "Entomopathogenic Nematodes in Biological Control", FLORIDA: CRC PRESS, pages 301-323. *

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US5908785A (en) 1999-06-01

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